- Email: [email protected]
Comparative study on brush border membranes prepared from rat and monkey small intestine by Ca2+ and Mg2+ precipitation
~
Comp. Biochem. Physiol. Vol. 112B, No. 1, pp. 65-69, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00
Pergamon
0305-0491(95)00057-7
Comparative study on brush border membranes prepared from rat and monkey small intestine by C a 2+ and Mg 2+ precipitation S. A. Ibrahim and K. A. Balasubramanian The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore-632 004, India Brush border membranes, prepared by Ca 2+ or Mg 2+ precipitation, from monkey or rat small intestine were compared for marker enzyme enrichment and lipid composition. Membranes prepared from rat intestine by Mg z+ precipitation were less pure in terms of their marker enzyme enrichment than Ca 2÷ precipitated membranes. Moreover, Mg 2+ precipitated membranes were contaminated by basolateral membranes as evidenced by the enrichment of Na+-K + ATPase. The content and composition of phospholipids were different in Mg 2+ and Ca 2÷ precipitated membranes. The membranes prepared by the two methods from monkeys were identical in their marker enzyme enrichment and lipid composition and unlike those obtained from the rat, there was no basolaterai membrane contamination. Key words: Rat; Monkey; Small intestine; Brush border membrane; Divalent cation precipitation;
Lipid composition. Comp. Biochem. Physiol. 112B, 65-69, 1995.
Introduction
A variety of techniques has been employed for the isolation of small intestinal brush border membranes. These include, cell disruption followed by density gradient centrifugation (Eichholz and Crane, 1965), immunoadsorbant chromatography (Carlson et al., 1982) and selective precipitation of other membranes using the divalent cations Ca 2+ (Schmitz et al., 1973; Kessler et al., 1978) or Mg z÷ (Hauser et al., 1980). The cation precipitation method is rapid and widely used. Subsequent treatment of the isolated brush border membranes with KCNS, selectively removed the cytoskeletal material and improved the fold purification (Hopfer et al., 1983). Brush border membranes prepared from rodent intestine by Ca 2+ and Mg 2+ precipitation differ in their lipid composition
(Hauser et al., 1980), size and shape, intramembranous particle distribution and protein composition (Bjorkman et al., 1986), phospholipid composition, and static and dynamic components of fluidity (Bjorkman and Brigham, 1990). It is not known whether these observations are applicable to the primate intestine. Since structural and functional aspects of brush border membranes are influenced by its lipid and protein composition (Proulx, 1991), it is important to isolate the membranes in the purest form with intact structural features. We studied the composition of rat and monkey small intestinal brush border membranes prepared by Ca 2+ and Mg 2+ precipitation in terms of the marker enzyme enrichment and lipid composition.
Correspondence to: K. A. Balasubramanian, The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore-632 004, India. Received 2 September 1994; revised 2 February 1995; accepted 16 February 1995.
M a t e r i a l s and M e t h o d s
Isolation of brush border membranes Rats weighing 150-200g, fasted overnight and were killed by cervical dislocation. The 65
66
s.A. Ibrahim and K. A. Balasubramanian
small intestine was excised, washed with ice-cold saline and the mucosa scraped off with a glass slide. Monkeys (Macacca radiata) fasted overnight were killed by a lethal dose of pentobarbitone. The small intestine was removed, washed with ice cold saline and the mucosa scraped with a glass slide. Brush border membranes were prepared by CaCI2 precipitation (Kessler et al., 1978). Briefly, the small intestinal mucosa was homogenized in 2 m M Tris/HC1 buffer (pH7.1) containing 5 0 m M mannitol; solid CaC12 was added to this to a final concentration of 10 m M , stirred for 1 min and allowed to stand at 4°C for 15rain. This was then centrifuged at 5000 g for 10 min and the supernatant was respun again at 48,000 g for 20 rain. The pellet obtained was suspended in 10 m M Hepes/Tris buffer containing 300 m M mannitol p H 7.5 using a syringe fitted with a 26 G needle. Alternatively brush border membranes were prepared by MgClz precipitation (Hauser et al., 1980). Protein was determined using bovine serum albumin as standard (Lowry et al., 1951). Activities of alkaline phosphatase (Thambidorai and Bachhawat, 1977), sucrase (Dahlqvist, 1968) and N a ÷ - K ÷ ATPase (Quigley and Gotterer, 1969) were measured in the isolated brush border membranes and homogenate.
Lipid analysis Lipids were extracted by the Bligh and Dyer (1959) method. Neutral lipids were separated on silica gel G plates using the solvent system hexane: diethylether: acetic acid (80: 20 : 1, v/v). Individual lipids were identified by iodine exposure, eluted with 3 ml c h l o r o f o r m : m e t h a n o l (2 : 1) thrice and evaporated to dryness using dry
nitrogen. Cholesterol (Zlatkis et al., 1953) and diglycerides and triglycerides (Snyder and Stephen, 1959) were estimated as described. Free fatty acids were separated and quantitated after methylation using gas chromatography. This was done using a Pye-Unicam PU4550 gas chromatograph equipped with a flame ionization detector and Spectraphysics PU4811 integrator. Fatty acids were separated on a 5% EGSS-X column and heptadecanoic acid was used as the internal standard. Phospholipids were separated on T L C using the solvent system chloroform: methanol:acetic acid :water (25: 15 : 4: 2, v/v) (Skipski et al., 1964). Spots were identified using authentic standards, eluted with chloroform: methanol: water (5:5:1, v/v) and quantitated by acid digestion followed by phosphate estimation (Bartlett, 1959). Aminophospholipids were separated and quantitated after fluorescamine derivatization (Schmid et al., 1981).
Results Table 1 shows the comparison of marker enzyme enrichment in brush border membranes isolated from rat intestine by Ca 2+ and Mg 2+ precipitation. With Ca 2+ precipitation method, the alkaline phosphatase and sucrase enrichment were 15.03- and 19.75-fold, respectively, whereas Mg 2+ precipitated membranes showed less enrichment in the final brush border membrane preparation. The specific activity of N a + - K ÷ ATPase which is present in basolateral membrane was increased nearly two-fold in the case of Mg 2+ precipitated membranes. This was not seen in Ca 2+ precipitated membranes.
Table 1. Comparison of marker enzymes of rat and monkey small intestinal brush border membranes prepared by Ca2+ and Mg2+ precipitation Alkaline Sucrase Na+-K + ATPase phosphatase (units/mg protein) Rat Mg 2+ precipitation Homogenate Brush border membrane Fold purification
0.21 __+0.01 2.46 + 0.24 I 1.71
0.04 + 0.01 0.59 __+0.12 14.75
1.07 +_ 0.19 2.52 + 0.17 2.35
Ca 2+ precipitation Homogenate Brush border membrane Fold purification
0.26 + 0.06 3.91 + 0.34 15.03
0.04 + 0.01 0.79 + 0.13 19.75
1.07 + 0.19 0.53 + 0.15 --
Monkey Mg 2+ precipitation Homogenate Brush border membrane Fold purification
0.10 __+0.01 1.08 + 0.33 10.80
0.05 + 0.01 0.78 ! 0.11 15.60
11.96 __+2.86 5.49 __+1.30 --
Ca 2+ precipitation Homogenate Brush border membrane Fold purification
0.10 _+ 0.02 1.24 + 0.36 12.40
0.05 + 0.05 0.82 + 0.11 16.40
12.70 __+2.24 4.93 __+1.20 --
Each value represents mean _+SD of three separate estimations.
Intestinal brush border membrane preparation Table 2. Comparison of lipid composition of rat and monkey small intestinal brush border membranes prepared by Ca2+ and Mg2+ precipitation Mg2+ C a 2+ precipitation precipitation (nmol/mg protein) Rat
Total phospholipids 365.4 + 22 257.3 + 14 Total cholesterol 198.1 +__4 164.0 +__12 Free fatty acids 179.5 ± 14 188.7 ± 10 Diglycerides 43.0 ± 2 32.0 ± 4 Triglycerides 42.3 ± 4 49.1 ± 6 C/P ratio 0.542 0.637 Monkey Total phospholipids 481.0 _ 28 499.0 +__26 Total cholesterol 237.2 +__17 234.1 + 29 Free fatty acids 210.6 ± 4 230.7 __+4 Diglycerides 30.3 ± 5 43.1 ± 8 Triglycerides 57.7 __+2 54.0 ___13 C/P ratio 0.493 0.469 Each value represents mean + SD of three separate estimation. C/P ratio, cholesterol/phospholipid ratio. Table 1 also shows the comparison of marker enzymes in brush border membranes isolated from monkey small intestine by Ca 2+ and Mg 2÷ precipitation and Table 2, the lipids. The enrichment of marker enzymes was identical irrespective of the cation used for precipitation. Specific activity of N a + - K ÷ ATPase was found to be decreased in the monkey brush border membranes prepared either by Ca 2+ or Mg 2÷ precipitation as compared to homogenate activity, although the activity of this enzyme was higher in the monkey intestinal brush border membrane as compared to that of rat. Brush border membranes prepared from rat small intestine had higher phospholipids in Mg 2÷ precipitated membranes as compared to Ca 2+ precipitated membranes and the content of other lipids was not significantly altered. In the case of monkey, there was no change in the lipid composition of the brush border membranes prepared by either Ca 2+ or Mg 2+ precipitation. The phospholipid composition of brush border membranes prepared by Ca 2÷ and Mg 2÷ precipitation from rat and monkey small intestine are shown in Fig. 1. The major phospholipids in the brush border membranes from both the species were phosphatidylethanolamine and phosphatidylcholine. In the case of rat, brush border membranes prepared by Ca 2+ precipitation had a significantly reduced level of phosphatidylethanolamine and to a lesser extent phosphatidylserine and phosphatidylinositol as compared to the Mg 2+ precipitated membrane. This reduction in phosphatidylethanolamine was not represented by an increase in lysophosphatidylethanolamine. Aminophospholipids were also separately quantitated after fluorescamine derivatization, a more specific and sensitive method which also showed a
67
decrease in phosphatidylethanolamine in the Ca 2+ precipitated brush border membrane from rat without any change in the level of lysophosphatidylethanolamine. As compared to rat, the phospholipid composition of monkey small intestinal brush border membranes remained the same in both Ca 2+ and Mg 2÷ precipitated membranes. Discussion
The enrichment of marker enzyme was different between Ca 2+ and Mg 2+ precipitated membranes in the case of rat but not in the monkey, suggesting that Mg 2÷ precipitated membranes from the rat intestine may be less pure. The enrichment of N a + - K ÷ ATPase in Mg 2÷ precipitated membranes from rat intestine indicates contamination by the basolateral membrane. A decrease in total phospholipids was observed in Ca 2+ precipitated membranes as compared to Mg 2÷ precipitated membranes and this may be due to activation of phospholipases by calcium ions during membrane preparation. Intestinal mucosal membranes are known to contain high phospholipase activity (Gallai-Hatchard and Thompson, 1965) but brush border membrane phospholipase is calcium independent and breakdown of phospholipids by phospholipase A 2 can occur even at - 2 0 ° C (Pind and Kuksis, 1987, 1988). Since the levels of lysophospholipids and free fatty acids in the two membrane preparations were comparable, metal ion activation of phospholipases may not be responsible for the observed decrease in the level of phospholipids. Several other reports have shown the presence of normal amounts of lysophospholipids in the brush border mere-
125 F Rat
-]- _
1-
ml Mg2+
50 ~" 25 o
LPC SM 175[-Monkey
PC PS+PI PE LPE TT 7-
125 1
~ 100 5 0 L j ~ 75 5O 25 0 LPC SM PC PS+PI PE
Fig. h Comparison of phospholipid composition of brush border membranes isolated from rat and monkey small intestine by Ca2+ or Mg2+ precipitation. Each value represents mean ± SD of three separate estimations.
68
s. A. Ibrahim and K. A. Balasubramanian
b r a n e s p r e p a r e d with C a 2÷ (Chapelle a n d GillesBaillien, 1983; A u b r y e t al., 1986). Different levels o f p h o s p h o l i p i d s in C a 2÷ a n d M g 2+ precipi t a t e d m e m b r a n e m a y be b r o u g h t a b o u t by p h o s p h o l i p a s e B activity a n d the presence o f this enzyme activity has been shown in intestinal brush border membranes (Grassama-Diagne e t al., 1989, 1992). This p h o s p h o l i p a s e is calc i u m - i n d e p e n d e n t a n d shows b o t h p h o s p h o lipase A2 a n d l y s o p h o s p h o l i p a s e activities resulting in the f o r m a t i o n o f free fatty acids w i t h o u t altering the l y s o p h o s p h o l i p i d content., I f p h o s p h o l i p a s e B is i n v o l v e d in a l t e r a t i o n o f p h o s p h o l i p i d c o n t e n t , one w o u l d expect a decrease in p h o s p h o l i p i d c o n t e n t in b o t h C a 2÷ a n d M g 2÷ p r e c i p i t a t e d m e m b r a n e s since this p h o s p h o l i p a s e is c a l c i u m - i n d e p e n d e n t . A similar c o n t e n t o f free fatty acids in b o t h the precipit a t i o n m e t h o d s suggests t h a t p h o s p h o l i p a s e B m a y n o t have a role in p h o s p h o l i p i d a l t e r a t i o n . Brush b o r d e r m e m b r a n e s r e p o r t e d f r o m rat by M g 2÷ p r e c i p i t a t i o n h a d a higher c o n t e n t o f phosphatidylethanolamine than membranes p r e p a r e d b y C a 2÷ p r e c i p i t a t i o n , a difference n o t seen in the b r u s h b o r d e r m e m b r a n e p r e p a r e d f r o m m o n k e y tissue. This difference in the lipid c o m p o s i t i o n f r o m rat tissue a p p e a r s to be due to c o n t a m i n a t i o n by the b a s o l a t e r a l m e m b r a n e . E a r l i e r studies have s h o w n t h a t b r u s h b o r d e r m e m b r a n e s p r e p a r e d f r o m rat a n d r a b b i t small intestinal m u c o s a b y M g 2÷ p r e c i p i t a t i o n were c o n t a m i n a t e d by b a s o l a t e r a l m e m b r a n e s ( A u b r y e t al., 1986; Stieger a n d M u r e r , 1983). It is also k n o w n t h a t the lipid c o m p o s i t i o n s o f b r u s h border membranes and basolateral membranes are different (Chapelle a n d Gilles-Baillien, 1983). B a s o l a t e r a l m e m b r a n e s have a higher level o f p h o s p h o l i p i d s a n d cholesterol, a n d the possible contamination by basolateral membranes in M g 2÷ p r e c i p i t a t i o n m e t h o d results in an increase in these lipids. A l s o b a s o l a t e r a l m e m b r a n e s are richer in p h o s p h a t i d y l e t h a n o l a m i n e phosphatidylserine and phosphatidylcholine t h a n b r u s h b o r d e r m e m b r a n e s . T h e increased levels o f p h o s p h a t i d y l e t h a n o l a m i n e a n d p h o s p h a t i d y l s e r i n e seen in M g 2÷ p r e c i p i t a t e d rat m e m b r a n e s further suggest c o n t a m i n a t i o n by basolateral membranes. In conclusion, this study s h o w e d species varia t i o n in the b r u s h b o r d e r m e m b r a n e s p r e p a r e d b y C a 2÷ a n d M g 2+ p r e c i p i t a t i o n . M e m b r a n e s p r e p a r e d f r o m m o n k e y h a d identical m a r k e r e n z y m e e n r i c h m e n t a n d lipid c o m p o s i t i o n irrespective o f m e t a l ions used, w h e r e a s C a 2÷ precipi t a t e d r a t m e m b r a n e s were m o r e p u r e in t e r m s o f m a r k e r e n z y m e e n r i c h m e n t a n d lipid c o m p o sition. Acknowledgements--This work was carried out in the Well-
come Trust Research Laboratory which is supported by The
Wellcome Trust, London. Financial assistance from the Indian Council of Medical Research, Government of India is acknowledged. The authors thank Prof. V. I. Mathan for his keen interest in this work and Prof. Anand Date for critically reading the manuscript.
References Aubry H., Merrill A. R. and Proulx P. (1986) A comparison of brush border membranes prepared from rabbit small intestine by procedures involving Ca 2+ and Mg2+ precipitation. Biochim. biophys. Acta. 856, 610-614. Bartlett G. R. (1959) Phosphorus assay in column chromatography. J. biol. Chem. 234, 466-468. Bjorkman D. J., Allan C. H., Hagen S. J. and Trier J. S. (1986) Structural features of absorptive cell and microvillus membrane preparations from rat small intestine. Gastroenterology 91, 1401-1414. Bjorkman D. J. and Brigham E. J. (1990) Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods. Biochem. biophys. Res. Commun. 170, 433-440. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. Carlsen J., Christiansen K. and Bro B. (1982) Purification of microvillus membrane vesicles from pig small intestine by immunoadsorbent chromatography. Biochim. biophys. Acta 689, 12-20. Chapelle S. and Gilles-Baillien M. (1983) Phospholipids and cholesterol in brush border and basolateral membrane from rat intestinal mucosa. Biochim. biophys. Acta. 759, 269-271. Dahlqvist A. (1968) Assay of intestinal disaccaridases. Analyt. Biochem. 22, 99-107. Eichholz A. and Crane R. K. (1965) Studies on the organization of the brush border in intestinal epithelial cells I. Tris disruption of isolated hamster brush borders and density gradient separation of fractions. J. cell. Biol. 26, 687-69. Gallai-Hatchard J. J. and Thompson R. H. S. (1965) Phospholipase-A activity of mammalian tissues. Biochim. biophys. Acta. 98, 128-132. Gassama-Diagne A., Fauvel J. and Chap H. (1989) Purification of a new calcium independent high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush border membrane. J. biol. Chem. 264, 9470-9475. Gassama-Diagne A., Rogalle P., Fauvel J., Willson M., Klaebe P. and Chap H. (1992) Substrate specificity of phospholipase B from guinea pig intestine. J. biol. Chem. 267, 13,418-13,424. Hauser H., Howell K., Dawson R. M. C. and Bowyer D. E. (1980) Rabbit small intestinal brush border membrane preparation and lipid composition. Biochim. biophys. Acta 602, 567 577. Hopfer U., Crowe T. D, and Tandler B. (1983) Purification of brush border membrane by thiocyanate treatment. Analyt. Biochem. 131, 447-452. Kessler M., Acuto O., Storelli C., Murer H., Muller M. and Semenza G. (1978) A modified procedure for rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim. biophys. Acta 506, 136154. Lowry O. H., Rosebrough N. J,, Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Pind S. and Kuksis A. (1987) Isolation of purified brush border membranes from rat jejunum containing a Ca2+ independent phospholipase A: activity. Biochim. biophys. Acta 901, 78-87.
Intestinal brush border membrane preparation Pind S. and Kuksis A. (1988) Solubilization and assay of phospholipase A2 activity from rat jejunal brush border membranes. Biochim. biophys. Acta 938, 211-221. Proulx P. (1991) Structure-function relationships in intestinal brush border membranes. Biochim. biophys. Acta 1071, 255-271. Quigley J. P. and Gotterer S. (3. (1969) Distribution o f N a ÷ K + ATPase activity in rat intestinal mucosa. Biochim. biophys. Acta 173, 456-468. Schmid P. C., Pfeiffer D. R. and Schmid H. H. O. (1981) Quantification of lysophosphatidylethanolamine in the nanomol range. J. Lipid Res. 22, 882-886. Schmitz J., Preiser H., Maestracci D., Ghosh B. K., Cerdar J. J. and Crane R. K. (1973) Purification of the human intestinal brush border membrane. Biochim. biophys..4cta 323, 98-112.
69
Skipski V. P., Peterson R. F. and Barclay M. (1964) Quantitative analysis of phospholipids by thin layer chromatography. Biochem. J. 90, 374-378. Snyder F. and Stephen N. (1959) A simplified spectrophotometric determination of ester groups in lipids. Biochim. biophys. Acta 34, 244-245. Stieger B. and Murer H. (1983) Hetrogenity of brush border membrane vesicles from rat small intestine prepared by precipitation method using Mg/EGTA. Eur. J. Biochem. 135, 95-101. Thambidorai D. and Bachhawat B. K. (1977) Purification and properties of brain alkaline phosphatase. J. Neurochem. 29, 503-512. Zlatkis A., Zak B. and Boyle A. J. (1953) A new method for the determination of serum cholesterol. J. Lab. clin. Med. 41, 486-492.
Comp. Biochem. Physiol. Vol. 112B, No. 1, pp. 65-69, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/95 $9.50 + 0.00
Pergamon
0305-0491(95)00057-7
Comparative study on brush border membranes prepared from rat and monkey small intestine by C a 2+ and Mg 2+ precipitation S. A. Ibrahim and K. A. Balasubramanian The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore-632 004, India Brush border membranes, prepared by Ca 2+ or Mg 2+ precipitation, from monkey or rat small intestine were compared for marker enzyme enrichment and lipid composition. Membranes prepared from rat intestine by Mg z+ precipitation were less pure in terms of their marker enzyme enrichment than Ca 2÷ precipitated membranes. Moreover, Mg 2+ precipitated membranes were contaminated by basolateral membranes as evidenced by the enrichment of Na+-K + ATPase. The content and composition of phospholipids were different in Mg 2+ and Ca 2÷ precipitated membranes. The membranes prepared by the two methods from monkeys were identical in their marker enzyme enrichment and lipid composition and unlike those obtained from the rat, there was no basolaterai membrane contamination. Key words: Rat; Monkey; Small intestine; Brush border membrane; Divalent cation precipitation;
Lipid composition. Comp. Biochem. Physiol. 112B, 65-69, 1995.
Introduction
A variety of techniques has been employed for the isolation of small intestinal brush border membranes. These include, cell disruption followed by density gradient centrifugation (Eichholz and Crane, 1965), immunoadsorbant chromatography (Carlson et al., 1982) and selective precipitation of other membranes using the divalent cations Ca 2+ (Schmitz et al., 1973; Kessler et al., 1978) or Mg z÷ (Hauser et al., 1980). The cation precipitation method is rapid and widely used. Subsequent treatment of the isolated brush border membranes with KCNS, selectively removed the cytoskeletal material and improved the fold purification (Hopfer et al., 1983). Brush border membranes prepared from rodent intestine by Ca 2+ and Mg 2+ precipitation differ in their lipid composition
(Hauser et al., 1980), size and shape, intramembranous particle distribution and protein composition (Bjorkman et al., 1986), phospholipid composition, and static and dynamic components of fluidity (Bjorkman and Brigham, 1990). It is not known whether these observations are applicable to the primate intestine. Since structural and functional aspects of brush border membranes are influenced by its lipid and protein composition (Proulx, 1991), it is important to isolate the membranes in the purest form with intact structural features. We studied the composition of rat and monkey small intestinal brush border membranes prepared by Ca 2+ and Mg 2+ precipitation in terms of the marker enzyme enrichment and lipid composition.
Correspondence to: K. A. Balasubramanian, The Wellcome Trust Research Laboratory, Department of Gastrointestinal Sciences, Christian Medical College & Hospital, Vellore-632 004, India. Received 2 September 1994; revised 2 February 1995; accepted 16 February 1995.
M a t e r i a l s and M e t h o d s
Isolation of brush border membranes Rats weighing 150-200g, fasted overnight and were killed by cervical dislocation. The 65
66
s.A. Ibrahim and K. A. Balasubramanian
small intestine was excised, washed with ice-cold saline and the mucosa scraped off with a glass slide. Monkeys (Macacca radiata) fasted overnight were killed by a lethal dose of pentobarbitone. The small intestine was removed, washed with ice cold saline and the mucosa scraped with a glass slide. Brush border membranes were prepared by CaCI2 precipitation (Kessler et al., 1978). Briefly, the small intestinal mucosa was homogenized in 2 m M Tris/HC1 buffer (pH7.1) containing 5 0 m M mannitol; solid CaC12 was added to this to a final concentration of 10 m M , stirred for 1 min and allowed to stand at 4°C for 15rain. This was then centrifuged at 5000 g for 10 min and the supernatant was respun again at 48,000 g for 20 rain. The pellet obtained was suspended in 10 m M Hepes/Tris buffer containing 300 m M mannitol p H 7.5 using a syringe fitted with a 26 G needle. Alternatively brush border membranes were prepared by MgClz precipitation (Hauser et al., 1980). Protein was determined using bovine serum albumin as standard (Lowry et al., 1951). Activities of alkaline phosphatase (Thambidorai and Bachhawat, 1977), sucrase (Dahlqvist, 1968) and N a ÷ - K ÷ ATPase (Quigley and Gotterer, 1969) were measured in the isolated brush border membranes and homogenate.
Lipid analysis Lipids were extracted by the Bligh and Dyer (1959) method. Neutral lipids were separated on silica gel G plates using the solvent system hexane: diethylether: acetic acid (80: 20 : 1, v/v). Individual lipids were identified by iodine exposure, eluted with 3 ml c h l o r o f o r m : m e t h a n o l (2 : 1) thrice and evaporated to dryness using dry
nitrogen. Cholesterol (Zlatkis et al., 1953) and diglycerides and triglycerides (Snyder and Stephen, 1959) were estimated as described. Free fatty acids were separated and quantitated after methylation using gas chromatography. This was done using a Pye-Unicam PU4550 gas chromatograph equipped with a flame ionization detector and Spectraphysics PU4811 integrator. Fatty acids were separated on a 5% EGSS-X column and heptadecanoic acid was used as the internal standard. Phospholipids were separated on T L C using the solvent system chloroform: methanol:acetic acid :water (25: 15 : 4: 2, v/v) (Skipski et al., 1964). Spots were identified using authentic standards, eluted with chloroform: methanol: water (5:5:1, v/v) and quantitated by acid digestion followed by phosphate estimation (Bartlett, 1959). Aminophospholipids were separated and quantitated after fluorescamine derivatization (Schmid et al., 1981).
Results Table 1 shows the comparison of marker enzyme enrichment in brush border membranes isolated from rat intestine by Ca 2+ and Mg 2+ precipitation. With Ca 2+ precipitation method, the alkaline phosphatase and sucrase enrichment were 15.03- and 19.75-fold, respectively, whereas Mg 2+ precipitated membranes showed less enrichment in the final brush border membrane preparation. The specific activity of N a + - K ÷ ATPase which is present in basolateral membrane was increased nearly two-fold in the case of Mg 2+ precipitated membranes. This was not seen in Ca 2+ precipitated membranes.
Table 1. Comparison of marker enzymes of rat and monkey small intestinal brush border membranes prepared by Ca2+ and Mg2+ precipitation Alkaline Sucrase Na+-K + ATPase phosphatase (units/mg protein) Rat Mg 2+ precipitation Homogenate Brush border membrane Fold purification
0.21 __+0.01 2.46 + 0.24 I 1.71
0.04 + 0.01 0.59 __+0.12 14.75
1.07 +_ 0.19 2.52 + 0.17 2.35
Ca 2+ precipitation Homogenate Brush border membrane Fold purification
0.26 + 0.06 3.91 + 0.34 15.03
0.04 + 0.01 0.79 + 0.13 19.75
1.07 + 0.19 0.53 + 0.15 --
Monkey Mg 2+ precipitation Homogenate Brush border membrane Fold purification
0.10 __+0.01 1.08 + 0.33 10.80
0.05 + 0.01 0.78 ! 0.11 15.60
11.96 __+2.86 5.49 __+1.30 --
Ca 2+ precipitation Homogenate Brush border membrane Fold purification
0.10 _+ 0.02 1.24 + 0.36 12.40
0.05 + 0.05 0.82 + 0.11 16.40
12.70 __+2.24 4.93 __+1.20 --
Each value represents mean _+SD of three separate estimations.
Intestinal brush border membrane preparation Table 2. Comparison of lipid composition of rat and monkey small intestinal brush border membranes prepared by Ca2+ and Mg2+ precipitation Mg2+ C a 2+ precipitation precipitation (nmol/mg protein) Rat
Total phospholipids 365.4 + 22 257.3 + 14 Total cholesterol 198.1 +__4 164.0 +__12 Free fatty acids 179.5 ± 14 188.7 ± 10 Diglycerides 43.0 ± 2 32.0 ± 4 Triglycerides 42.3 ± 4 49.1 ± 6 C/P ratio 0.542 0.637 Monkey Total phospholipids 481.0 _ 28 499.0 +__26 Total cholesterol 237.2 +__17 234.1 + 29 Free fatty acids 210.6 ± 4 230.7 __+4 Diglycerides 30.3 ± 5 43.1 ± 8 Triglycerides 57.7 __+2 54.0 ___13 C/P ratio 0.493 0.469 Each value represents mean + SD of three separate estimation. C/P ratio, cholesterol/phospholipid ratio. Table 1 also shows the comparison of marker enzymes in brush border membranes isolated from monkey small intestine by Ca 2+ and Mg 2÷ precipitation and Table 2, the lipids. The enrichment of marker enzymes was identical irrespective of the cation used for precipitation. Specific activity of N a + - K ÷ ATPase was found to be decreased in the monkey brush border membranes prepared either by Ca 2+ or Mg 2÷ precipitation as compared to homogenate activity, although the activity of this enzyme was higher in the monkey intestinal brush border membrane as compared to that of rat. Brush border membranes prepared from rat small intestine had higher phospholipids in Mg 2÷ precipitated membranes as compared to Ca 2+ precipitated membranes and the content of other lipids was not significantly altered. In the case of monkey, there was no change in the lipid composition of the brush border membranes prepared by either Ca 2+ or Mg 2+ precipitation. The phospholipid composition of brush border membranes prepared by Ca 2÷ and Mg 2÷ precipitation from rat and monkey small intestine are shown in Fig. 1. The major phospholipids in the brush border membranes from both the species were phosphatidylethanolamine and phosphatidylcholine. In the case of rat, brush border membranes prepared by Ca 2+ precipitation had a significantly reduced level of phosphatidylethanolamine and to a lesser extent phosphatidylserine and phosphatidylinositol as compared to the Mg 2+ precipitated membrane. This reduction in phosphatidylethanolamine was not represented by an increase in lysophosphatidylethanolamine. Aminophospholipids were also separately quantitated after fluorescamine derivatization, a more specific and sensitive method which also showed a
67
decrease in phosphatidylethanolamine in the Ca 2+ precipitated brush border membrane from rat without any change in the level of lysophosphatidylethanolamine. As compared to rat, the phospholipid composition of monkey small intestinal brush border membranes remained the same in both Ca 2+ and Mg 2÷ precipitated membranes. Discussion
The enrichment of marker enzyme was different between Ca 2+ and Mg 2+ precipitated membranes in the case of rat but not in the monkey, suggesting that Mg 2÷ precipitated membranes from the rat intestine may be less pure. The enrichment of N a + - K ÷ ATPase in Mg 2÷ precipitated membranes from rat intestine indicates contamination by the basolateral membrane. A decrease in total phospholipids was observed in Ca 2+ precipitated membranes as compared to Mg 2÷ precipitated membranes and this may be due to activation of phospholipases by calcium ions during membrane preparation. Intestinal mucosal membranes are known to contain high phospholipase activity (Gallai-Hatchard and Thompson, 1965) but brush border membrane phospholipase is calcium independent and breakdown of phospholipids by phospholipase A 2 can occur even at - 2 0 ° C (Pind and Kuksis, 1987, 1988). Since the levels of lysophospholipids and free fatty acids in the two membrane preparations were comparable, metal ion activation of phospholipases may not be responsible for the observed decrease in the level of phospholipids. Several other reports have shown the presence of normal amounts of lysophospholipids in the brush border mere-
125 F Rat
-]- _
1-
ml Mg2+
50 ~" 25 o
LPC SM 175[-Monkey
PC PS+PI PE LPE TT 7-
125 1
~ 100 5 0 L j ~ 75 5O 25 0 LPC SM PC PS+PI PE
Fig. h Comparison of phospholipid composition of brush border membranes isolated from rat and monkey small intestine by Ca2+ or Mg2+ precipitation. Each value represents mean ± SD of three separate estimations.
68
s. A. Ibrahim and K. A. Balasubramanian
b r a n e s p r e p a r e d with C a 2÷ (Chapelle a n d GillesBaillien, 1983; A u b r y e t al., 1986). Different levels o f p h o s p h o l i p i d s in C a 2÷ a n d M g 2+ precipi t a t e d m e m b r a n e m a y be b r o u g h t a b o u t by p h o s p h o l i p a s e B activity a n d the presence o f this enzyme activity has been shown in intestinal brush border membranes (Grassama-Diagne e t al., 1989, 1992). This p h o s p h o l i p a s e is calc i u m - i n d e p e n d e n t a n d shows b o t h p h o s p h o lipase A2 a n d l y s o p h o s p h o l i p a s e activities resulting in the f o r m a t i o n o f free fatty acids w i t h o u t altering the l y s o p h o s p h o l i p i d content., I f p h o s p h o l i p a s e B is i n v o l v e d in a l t e r a t i o n o f p h o s p h o l i p i d c o n t e n t , one w o u l d expect a decrease in p h o s p h o l i p i d c o n t e n t in b o t h C a 2÷ a n d M g 2÷ p r e c i p i t a t e d m e m b r a n e s since this p h o s p h o l i p a s e is c a l c i u m - i n d e p e n d e n t . A similar c o n t e n t o f free fatty acids in b o t h the precipit a t i o n m e t h o d s suggests t h a t p h o s p h o l i p a s e B m a y n o t have a role in p h o s p h o l i p i d a l t e r a t i o n . Brush b o r d e r m e m b r a n e s r e p o r t e d f r o m rat by M g 2÷ p r e c i p i t a t i o n h a d a higher c o n t e n t o f phosphatidylethanolamine than membranes p r e p a r e d b y C a 2÷ p r e c i p i t a t i o n , a difference n o t seen in the b r u s h b o r d e r m e m b r a n e p r e p a r e d f r o m m o n k e y tissue. This difference in the lipid c o m p o s i t i o n f r o m rat tissue a p p e a r s to be due to c o n t a m i n a t i o n by the b a s o l a t e r a l m e m b r a n e . E a r l i e r studies have s h o w n t h a t b r u s h b o r d e r m e m b r a n e s p r e p a r e d f r o m rat a n d r a b b i t small intestinal m u c o s a b y M g 2÷ p r e c i p i t a t i o n were c o n t a m i n a t e d by b a s o l a t e r a l m e m b r a n e s ( A u b r y e t al., 1986; Stieger a n d M u r e r , 1983). It is also k n o w n t h a t the lipid c o m p o s i t i o n s o f b r u s h border membranes and basolateral membranes are different (Chapelle a n d Gilles-Baillien, 1983). B a s o l a t e r a l m e m b r a n e s have a higher level o f p h o s p h o l i p i d s a n d cholesterol, a n d the possible contamination by basolateral membranes in M g 2÷ p r e c i p i t a t i o n m e t h o d results in an increase in these lipids. A l s o b a s o l a t e r a l m e m b r a n e s are richer in p h o s p h a t i d y l e t h a n o l a m i n e phosphatidylserine and phosphatidylcholine t h a n b r u s h b o r d e r m e m b r a n e s . T h e increased levels o f p h o s p h a t i d y l e t h a n o l a m i n e a n d p h o s p h a t i d y l s e r i n e seen in M g 2÷ p r e c i p i t a t e d rat m e m b r a n e s further suggest c o n t a m i n a t i o n by basolateral membranes. In conclusion, this study s h o w e d species varia t i o n in the b r u s h b o r d e r m e m b r a n e s p r e p a r e d b y C a 2÷ a n d M g 2+ p r e c i p i t a t i o n . M e m b r a n e s p r e p a r e d f r o m m o n k e y h a d identical m a r k e r e n z y m e e n r i c h m e n t a n d lipid c o m p o s i t i o n irrespective o f m e t a l ions used, w h e r e a s C a 2÷ precipi t a t e d r a t m e m b r a n e s were m o r e p u r e in t e r m s o f m a r k e r e n z y m e e n r i c h m e n t a n d lipid c o m p o sition. Acknowledgements--This work was carried out in the Well-
come Trust Research Laboratory which is supported by The
Wellcome Trust, London. Financial assistance from the Indian Council of Medical Research, Government of India is acknowledged. The authors thank Prof. V. I. Mathan for his keen interest in this work and Prof. Anand Date for critically reading the manuscript.
References Aubry H., Merrill A. R. and Proulx P. (1986) A comparison of brush border membranes prepared from rabbit small intestine by procedures involving Ca 2+ and Mg2+ precipitation. Biochim. biophys. Acta. 856, 610-614. Bartlett G. R. (1959) Phosphorus assay in column chromatography. J. biol. Chem. 234, 466-468. Bjorkman D. J., Allan C. H., Hagen S. J. and Trier J. S. (1986) Structural features of absorptive cell and microvillus membrane preparations from rat small intestine. Gastroenterology 91, 1401-1414. Bjorkman D. J. and Brigham E. J. (1990) Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods. Biochem. biophys. Res. Commun. 170, 433-440. Bligh E. G. and Dyer W. J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917. Carlsen J., Christiansen K. and Bro B. (1982) Purification of microvillus membrane vesicles from pig small intestine by immunoadsorbent chromatography. Biochim. biophys. Acta 689, 12-20. Chapelle S. and Gilles-Baillien M. (1983) Phospholipids and cholesterol in brush border and basolateral membrane from rat intestinal mucosa. Biochim. biophys. Acta. 759, 269-271. Dahlqvist A. (1968) Assay of intestinal disaccaridases. Analyt. Biochem. 22, 99-107. Eichholz A. and Crane R. K. (1965) Studies on the organization of the brush border in intestinal epithelial cells I. Tris disruption of isolated hamster brush borders and density gradient separation of fractions. J. cell. Biol. 26, 687-69. Gallai-Hatchard J. J. and Thompson R. H. S. (1965) Phospholipase-A activity of mammalian tissues. Biochim. biophys. Acta. 98, 128-132. Gassama-Diagne A., Fauvel J. and Chap H. (1989) Purification of a new calcium independent high molecular weight phospholipase A2/lysophospholipase (phospholipase B) from guinea pig intestinal brush border membrane. J. biol. Chem. 264, 9470-9475. Gassama-Diagne A., Rogalle P., Fauvel J., Willson M., Klaebe P. and Chap H. (1992) Substrate specificity of phospholipase B from guinea pig intestine. J. biol. Chem. 267, 13,418-13,424. Hauser H., Howell K., Dawson R. M. C. and Bowyer D. E. (1980) Rabbit small intestinal brush border membrane preparation and lipid composition. Biochim. biophys. Acta 602, 567 577. Hopfer U., Crowe T. D, and Tandler B. (1983) Purification of brush border membrane by thiocyanate treatment. Analyt. Biochem. 131, 447-452. Kessler M., Acuto O., Storelli C., Murer H., Muller M. and Semenza G. (1978) A modified procedure for rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim. biophys. Acta 506, 136154. Lowry O. H., Rosebrough N. J,, Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Pind S. and Kuksis A. (1987) Isolation of purified brush border membranes from rat jejunum containing a Ca2+ independent phospholipase A: activity. Biochim. biophys. Acta 901, 78-87.
Intestinal brush border membrane preparation Pind S. and Kuksis A. (1988) Solubilization and assay of phospholipase A2 activity from rat jejunal brush border membranes. Biochim. biophys. Acta 938, 211-221. Proulx P. (1991) Structure-function relationships in intestinal brush border membranes. Biochim. biophys. Acta 1071, 255-271. Quigley J. P. and Gotterer S. (3. (1969) Distribution o f N a ÷ K + ATPase activity in rat intestinal mucosa. Biochim. biophys. Acta 173, 456-468. Schmid P. C., Pfeiffer D. R. and Schmid H. H. O. (1981) Quantification of lysophosphatidylethanolamine in the nanomol range. J. Lipid Res. 22, 882-886. Schmitz J., Preiser H., Maestracci D., Ghosh B. K., Cerdar J. J. and Crane R. K. (1973) Purification of the human intestinal brush border membrane. Biochim. biophys..4cta 323, 98-112.
69
Skipski V. P., Peterson R. F. and Barclay M. (1964) Quantitative analysis of phospholipids by thin layer chromatography. Biochem. J. 90, 374-378. Snyder F. and Stephen N. (1959) A simplified spectrophotometric determination of ester groups in lipids. Biochim. biophys. Acta 34, 244-245. Stieger B. and Murer H. (1983) Hetrogenity of brush border membrane vesicles from rat small intestine prepared by precipitation method using Mg/EGTA. Eur. J. Biochem. 135, 95-101. Thambidorai D. and Bachhawat B. K. (1977) Purification and properties of brain alkaline phosphatase. J. Neurochem. 29, 503-512. Zlatkis A., Zak B. and Boyle A. J. (1953) A new method for the determination of serum cholesterol. J. Lab. clin. Med. 41, 486-492.